Biological applications of quantum dots

ABSTRACT

The present invention provides a composition comprising fluorescent semiconductor nanocrystals associated to a compound, wherein the nanocrystals have a characteristic spectral emission, wherein said spectral emission is tunable to a desired wavelength by controlling the size of the nanocrystal, and wherein said emission provides information about a biological state or event.

This application is a divisional of U.S. Ser. No. 09/160,454, filed onSep. 24, 1998, now U.S. Pat. No. 6,326,144 which claims priority fromprovisional application U.S. Ser. No. 60/100,947, filed on Sep. 18,1998, now abandoned, the entire contents of which are herebyincorporated by reference.

This application is related to the following applications that have beenincorporated in their entirety by reference: Application U.S. Ser. No.09/160,458 entitled “Inventory Control” filed Sep. 24, 1998 andapplication U.S. Ser. No. 09/156,863 entitled “Water-Soluble LuminescentNanocrystals” filed on Sep. 18, 1998.

This invention was made with government support under Grant No.DMR-9400334 awarded by the National Science Foundation. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to a composition comprising fluorescentsemiconductor nanocrystals associated with compounds for use inbiological applications.

BACKGROUND OF THE INVENTION

Traditional methods for detecting biological compounds in vivo and invitro rely on the use of radioactive markers. For example, these methodscommonly use radiolabeled probes such as nucleic acids labeled with ³²Por ³⁵S and proteins labeled with ³⁵S or ¹²⁵I to detect biologicalmolecules. These labels are effective because of the high degree ofsensitivity for the detection of radioactivity. However, many basicdifficulties exist with the use of radioisotopes. Such problems includethe need for specially trained personnel, general safety issues whenworking with radioactivity, inherently short half-lives with manycommonly used isotopes, and disposal problems due to full landfills andgovernmental regulations. As a result, current efforts have shifted toutilizing non-radioactive methods of detecting biological compounds.These methods often consist of the use of fluorescent molecules as tags(e.g. fluorescein, ethidium, methyl coumarin, rhodamine, and Texas red),or the use of chemiluminescence as a method of detection. Presentlyhowever, problems still exist when using these fluorescent andchemiluminescent markers. These problems include photobleaching,spectral separation, low fluorescence intensity, short half-lives, broadspectral linewidths, and non-gaussian asymmetric emission spectra havinglong tails.

Fluorescence is the emission of light resulting from the absorption ofradiation at one wavelength (excitation) followed by nearly immediatereradiation usually at a different wavelength (emission). Fluorescentdyes are frequently used as tags in biological systems. For example,compounds such as ethidium bromide, propidium iodide, Hoechst dyes, andDAPI (4′,6-diamindino-2-phenylindole) interact with DNA and fluoresce tovisualize DNA. Other biological components can be visualized byfluorescence using techniques such as immunofluorescence which utilizesantibodies labeled with a fluorescent tag and directed at a particularcellular target. For example, monoclonal or polyclonal antibodies taggedwith fluorescein or rhodamine can be directed to a desired cellulartarget and observed by fluorescence microscopy. An alternate method usessecondary antibodies that are tagged with a fluorescent marker anddirected to the primary antibodies to visualize the target.

Another application of fluorescent markers to detect biologicalcompounds is fluorescence in situ hybridization (FISH). This methodinvolves the fluorescent tagging of an oligonucleotide probe to detect aspecific complementary DNA or RNA sequence. An alternative approach isto use an oligonucleotide probe conjugated with an antigen such asbiotin or digoxygenin and a fluorescently tagged antibody directedtoward that antigen to visualize the hybridization of the probe to itsDNA target. FISH is a powerful tool for the chromosomal localization ofgenes whose sequences are partially or fully known. Other applicationsof FISH include in situ localization of mRNA in tissues samples andlocalization of non-genetic DNA sequences such as telomeres.

Fluorescent dyes also have applications in non-cellular biologicalsystems. For example, the advent of fluorescently-labeled nucleotideshas facilitated the development of new methods of high-throughput DNAsequencing and DNA fragment analysis (ABI system; Perkin-Elmer, Norwalk,Conn.). DNA sequencing reactions that once occupied four lanes on DNAsequencing gels can now be analyzed simultaneously in one lane. Briefly,four reactions are performed to determine the positions of the fournucleotide bases in a DNA sequence. The DNA products of the fourreactions are resolved by size using polyacrylamide gel electrophoresis.With singly radiolabeled (³²P or ³⁵S) DNA, each reaction is loaded intoan individual lane. The resolved products result in a pattern of bandsthat indicate the identity of a base at each nucleotide position. Thispattern across four lanes can be read like a simple code correspondingto the nucleotide base sequence of the DNA template. With fluorescentdideoxynucleotides, samples containing all four reactions can be loadedinto a single lane. Resolution of the products is possible because eachsample is marked with a different colored fluorescent dideoxynucleotide.For example, the adenine sequencing reaction can be marked with a greenfluorescent tag and the other three reactions marked with differentfluorescent colors. When all four reactions are analyzed in one lane ona DNA sequencing gel, the result is a ladder of bands consisting of fourdifferent colors. Each fluorescent color corresponds to the identity ofa nucleotide base and can be easily analyzed by automated systems.

There are chemical and physical limitations to the use of organicfluorescent dyes. One of these limitations is the variation ofexcitation wavelengths of different colored dyes. As a result,simultaneously using two or more fluorescent tags with differentexcitation wavelengths requires multiple excitation light sources. Thisrequirement thus adds to the cost and complexity of methods utilizingmultiple fluorescent dyes.

Another drawback when using organic dyes is the deterioration offluorescence intensity upon prolonged exposure to excitation light. Thisfading is called photobleaching and is dependent on the intensity of theexcitation light and the duration of the illumination. In addition,conversion of the dye into a nonfluorescent species is irreversible.Furthermore, the degradation products of dyes are organic compoundswhich may interfere with biological processes being examined.

Another drawback of organic dyes is the spectral overlap that existsfrom one dye to another. This is due in part to the relatively wideemission spectra of organic dyes and the overlap of the spectra near thetailing region. Few low molecular weight dyes have a combination of alarge Stokes shift, which is defined as the separation of the absorptionand emission maxima, and high fluorescence output. In addition, lowmolecular weight dyes may be impractical for some applications becausethey do not provide a bright enough fluorescent signal. The idealfluorescent label should fulfill many requirements. Among the desiredqualities are the following: (i) high fluorescent intensity (fordetection in small quantities), (ii) a separation of at least 50 nmbetween the absorption and fluorescing frequencies, (iii) solubility inwater, (iv) ability to be readily linked to other molecules, (v)stability towards harsh conditions and high temperatures, (vi) asymmetric, nearly gaussian emission lineshape for easy deconvolution ofmultiple colors, and (vii) compatibility with automated analysis. Atpresent, none of the conventional fluorescent labels satisfies all theserequirements. Furthermore, the differences in the chemical properties ofstandard organic fluorescent dyes make multiple, parallel assays quiteimpractical since different chemical reactions may be involved for eachdye used in the variety of applications of fluorescent labels.

SUMMARY OF THE INVENTION

The present invention provides a composition that can provideinformation about a biological state or event. The composition by way ofexample can detect the presence or amounts of a biological moiety; thestructure, composition, and conformation of a biological moiety; thelocalization of a biological moiety in an environment; interactions ofbiological moieties; alterations in structures of biological compounds;and alterations in biological processes.

The composition is comprised of a fluorescent semiconductor nanocrystal(known as a quantum dot) having a characteristic spectral emission,which is tunable to a desired energy by selection of the particle sizeof the quantum dot. The composition further comprises a compound,associated with the quantum dot that has an affinity for a biologicaltarget. The composition interacts or associates with a biological targetdue to the affinity of the compound with the target. Location and natureof the association can be detected by monitoring the emission of thequantum dot.

In operation, the composition is introduced into an environmentcontaining a biological target and the composition associates with thetarget. The composition:target complex may be spectroscopically viewedby irradiation of the complex with an excitation light source. Thequantum dot emits a characteristic emission spectrum which can beobserved and measured spectrophotometrically.

As an advantage of the composition of the present invention, theemission spectra of quantum dots have linewidths as narrow as 25-30 nmdepending on the size heterogeneity of the sample, and lineshapes thatare symmetric, gaussian or nearly gaussian with an absence of a tailingregion. The combination of tunability, narrow linewidths, and symmetricemission spectra without a tailing region provides for high resolutionof multiply-sized quantum dots within a system and enables researchersto examine simultaneously a variety of biological moieties tagged withQDs.

In addition, the range of excitation wavelengths of the nanocrystalquantum dots is broad and can be higher in energy than the emissionwavelengths of all available quantum dots. Consequently, this allows thesimultaneous excitation of all quantum dots in a system with a singlelight source, usually in the ultraviolet or blue region of the spectrum.QDs are also more robust than conventional organic fluorescent dyes andare more resistant to photobleaching than the organic dyes. Therobustness of the QD also alleviates the problem of contamination of thedegradation products of the organic dyes in the system being examined.Therefore, the present invention provides uniquely valuable tags fordetection of biological molecules and the interactions they undergo.

In one preferred embodiment, the composition comprises quantum dotsassociated with molecules that can physically interact with biologicalcompounds. Without limiting the scope of the invention, moleculesinclude ones that can bind to proteins, nucleic acids, cells,subcellular organelles, and other biological molecules. The compoundused in the composition of the present invention preferably has anaffinity for a biological target. In some preferred embodiments, thecompound has a specific affinity for a biological target. The affinitymay be based upon any inherent properties of the compound, such aswithout limitation, van der Waals attraction, hydrophilic attractions,ionic, covalent, electrostatic or magetic attraction of the compound toa biological target. As used herein, “biological target” is meant anymoiety, compound, cellular or sub-cellular component which is associatedwith biological functions. The biological target includes withoutlimitation proteins, nucleic acids, cells, subcellular organelles andother biological moieties.

In another preferred embodiment, the composition comprises quantum dotsassociated with proteins. Without limiting the scope of the invention,the proteins may be antibodies that are directed towards specificbiological antigens such as other proteins, nucleic acids, subcellularorganelles, and small molecules that are conjugated to biologicalcompounds. The proteins may also be proteins that interact specificallyor non-specifically with other biological compounds.

In another preferred embodiment, the composition comprises quantum dotsassociated with nucleic acids. Without limiting the scope of theinvention, the nucleic acids may be oligonucleotides ordeoxyribooligonucleotides that hybridize to nucleic acid polymers invivo or in vitro. The nucleic acids may also be nucleotides,deoxynucleotides, dideoxynucleotides, or derivatives and combinationsthereof that are used for the synthesis of DNA or RNA.

In another aspect of the invention, a method of detecting biologicalcompounds using quantum dots is provided.

DEFINITIONS

-   -   Quantum dots are a semiconductor nanocrystal with size-dependent        optical and electronic properties. In particular, the band gap        energy of a quantum dot varies with the diameter of the crystal.    -   DNA is deoxyribonucleic acid.    -   Monodispersed particles are defined as having at least 60% of        the particles fall within a specified particle size range.        Monodispersed particles deviate less than 10% in rms diameter        and preferably less than 5%.    -   Quantum yield is defined as the ratio of photons emitted to that        absorbed.    -   A small molecule is defined as an organic compound either        synthesized in the laboratory or found in nature. Typically, a        small molecule is characterized in that it contains several        carbon-carbon bonds, and has a molecular weight of less than        1500 grams/Mol    -   A biological state is defined as the quantitative and        qualitative presence of a biological moiety; the structure,        composition, and conformation of a biological moiety; and the        localization of a biological moiety in an environment.    -   Biological events are defined as interactions of biological        moieties, biological processes, alterations in structures of        biological compounds, and alterations in biological processes.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a cartoon depiction of the single-sized quantum dotpreparation labeled immunoassay.

FIG. 2 is a cartoon depiction of the multicolored quantum dot labeled,parallel immunoassay.

FIG. 3 is a cartoon depiction of the use of two differently colored QDsor one color QD and one organic dye to detect proximity of compounds. Inthis example, two oligonucleotide probes are hybridized to DNA sequencesin close proximity and detected by fluorescence resonance energytransfer

FIG. 4 is a cartoon depiction of the formation of water-soluble quantumdots by cap exchange

FIG. 5 outlines the reaction between biotin and hexane dithiol to formthe biotin-hexane dithiol (BHDT) derivative.

FIG. 6 outlines the reaction between biotin and a diamine to formbiotin-amine derivative.

FIG. 7 depicts the formation of the biotin-thiol-dot complex for awater-soluble dot.

FIG. 8 depicts the formation of the biotin-amine-dot complex where theamine is adsorbed to the outer layer of the dot.

FIG. 9 depicts the formation of the biotin-amine-dot complex where theamine is conjugated to the carboxylic acid group of thewater-solubilizing later.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a composition comprising semiconductornanocrystals (also referred to in this application as quantum dots; QDs)as fluorescent tags associated with a reagent or molecule wherein thecomposition can detect the presence or amount of a biological molecule,detect biological interactions, detect biological processes, detectalterations in biological processes, or detect alteractions in thestructure of a biological compound.

Semiconductor nanocrystals (quantum dots) demonstrate quantumconfinement effects in their luminescent properties. When quantum dotsare illuminated with a primary energy source, a secondary emission ofenergy occurs of a frequency that corresponds to the band gap of thesemiconductor material used in the quantum dot. In quantum confinedparticles, the band gap is a function of the size of the nanocrystal.

Many semiconductors that are constructed of elements from groups II-VI,III-V and IV of the periodic table have been prepared as quantum sizedparticles, exhibit quantum confinement effects in their physicalproperties, and can be used in the composition of the invention.Exemplary materials suitable for use as quantum dots include ZnS, ZnSe,ZnTe, CdS, CdSe, CdTe, GaN, GaP, GaAs, GaSb, InP, InAs, InSb, AlS, AlP,AlAs, AlSb, PbS, PbSe, Ge, and Si and ternary and quaternary mixturesthereof. The quantum dots may further include an overcoating layer of asemiconductor having a greater band gap.

The semiconductor nanocrystals are characterized by their uniformnanometer size. By “nanometer” size, it is meant less than about 150Angstroms (Å), and preferably in the range of 12-150 Å. The nanocrystalsalso are substantially monodisperse within the broad nanometer rangegiven above. By monodisperse, as that term is used herein, it is meant acolloidal system in which the suspended particles have substantiallyidentical size and shape. For the purposes of the present invention,monodisperse particles mean that at least 60% of the particles fallwithin a specified particle size range. Monodisperse particles deviateless than 10% in rms diameter, and preferably less than 5%.

The narrow size distribution of the quantum dots allows the possibilityof light emission in narrow spectral widths. Monodisperse quantum dotshave been described in detail in Murray et al. (J. Am. Chem. Soc.,115:8706 (1993)); in the thesis of Christopher Murray, “Synthesis andCharacterization of II-VI Quantum Dots and Their Assembly into 3-DQuantum Dot Superlattices”, Massachusetts Institute of Technology,September, 1995; and in U.S. patent application Ser. No. 08/969,302entitled “Highly Luminescent Color-selective Materials” which are herebyincorporated in their entireties by reference.

The fluorescence of semiconductor nanocrystals results from confinementof electronic excitations to the physical dimensions of thenanocrystals. In contrast to the bulk semiconductor material from whichthese dots are synthesized, these quantum dots have discrete opticaltransitions, which are tunable with size (U.S. patent application Ser.No. 08/969,302 entitled “Highly Luminescent Color-selective Materials”).Current technology allows good control of their sizes (between 12 to 150Å; standard deviations approximately 5%), and thus, enables constructionof QDs that emit light at a desired wavelength throughout theUV-visible-IR spectrum with a quantum yield ranging from 30-50% at roomtemperature in organic solvents and 10-30% at room temperature in water.

The present invention provides a composition comprising semiconductornanocrystals (quantum dots) associated with a reagent or molecule suchthat the composition can detect the presence and amounts of biologicalcompounds, detect interactions in biological systems, detect biologicalprocesses, detect alterations in biological processes, or detectalterations in the structure of biological compounds. Without limitationto the present invention, these reagents or molecules include anymolecule or molecular complex that can interact with a biologicaltarget, molecules or molecular complexes that can associate withbiological targets to detect biological processes, or reactions, andmolecules or molecular complexes that can alter biological molecules orprocesses. Preferably, the molecules or molecular complexes physicallyinteract with biological compounds. Preferably, the interactions arespecific. The interactions can be, but are not limited to, covalent,noncovalent, hydrophobic, hydrophilic, electrostatic, van der Waals, ormagnetic. Preferably, these molecules are small molecules, proteins, ornucleic acids or combinations thereof.

Quantum dots (QDs) are capable of fluorescence when excited by light.Currently, detection of biological compounds by photoluminescenceutilizes fluorescent organic dyes and chemiluminescent compounds. Theuse of QDs as fluorescent markers in biological systems providesadvantages over existing fluorescent organic dyes. Many of theseadvantages relate to the spectral properties of QDs. For example withoutlimiting the scope of the present invention, the ability to control thesize of QDs enables one to construct QDs with fluorescent emissions atany wavelength in the UV-visible-IR region. Therefore, the colors(emissions) of QDs are tunable to any desired spectral wavelength.Furthermore, the emission spectra of monodisperse QDs have linewidths asnarrow as 25-30 nm. The linewidths are dependent on the sizeheterogeneity of QDs in each preparation. Single quantum dots have beenobserved to have FWHMs of 12-15 nm. In addition, QDs with larger FWHM inthe range of 40-60 nm can be readily made and have the same physicalcharacteristics, such as emission wavelength tunability and excitationin the UV-blue, preferably in the blue region of the spectrum, as QDswith narrower FWHM.

The narrow spectral linewidths and nearly gaussian symmetricallineshapes lacking a tailing region observed for the emission spectra ofQDs combined with the tunability of the emission wavelengths of QDsallows high spectral resolution in a system with multiple QDs. Intheory, up to 10-20 different-sized QDs from different preparations ofQDs, with each sample having a different emission spectrum, can be usedsimultaneously in one system with the overlapping spectra easilyresolved using deconvolution software.

Another advantage to the use of QDs as fluorescent markers over existingorganic fluorescent dyes is that only a single light source (usually inthe UV-blue, and preferably in the blue region of the spectrum) isneeded to excited all QDs in a system. Organic dyes with differentemission wavelengths usually have different excitation wavelengths.Thus, multiple light sources or a single light source with adaptablelight-filters are needed for systems that utilize organic dyes withdifferent excitation wavelength. Since all QDs of the present inventioncan be excited by light in the UV-blue region of the spectrum(preferably the blue visible region), a single light source can be used.This minimizes the technical complexity needed to provide an excitationlight source. In addition, by using blue light, the source radiationwill not interfere with any of the fluorescence measurements taken inthe visible or infrared region of the light spectrum, and also will notdamage biological molecules. For example, UV light can causedimerization in DNA molecules.

Another advantage of the use of QDs over organic fluorescent dyes thatare currently available is the robust nature of the QDs due to theircrystalline inorganic structure and their protective overcoating layer.These QDs are more resistant to photobleaching than what is observed fororganic dyes. Also, since QDs described in the application are composedof similar materials and are protected by the same organic cappinggroups, chemical uniformity of QDs allows the extrapolation of aprotocol developed to attach one particular size of QDs to a molecule toQDs of all sizes within that class of QDs. Such uniformity should bevaluable in extending conventional assaying techniques to includeparallel detection schemes. Therefore, the present invention provides aseries of fluorescent probes, which span the spectrum from the UV to theIR, and also can have substantially identical chemical properties.

Because detection of biological compounds is most preferably carried outin aqueous media, a preferred embodiment of the present invention,utilizes quantum dots that are solubilized in water. Quantum dotsdescribed by Bawendi et al. (J. Am. Chem. Soc., 115:8706, 1993) aresoluble or dispersible only in organic solvents, such as hexane orpyridine. It is preferred that the QDs are water-soluble and associatedwith molecules capable of interacting with biological compounds.However,

construction of water-soluble QDs suitable for biological systems (U.S.Patent Application entitled “Water-Soluble Luminescent Nanocrystals”incorporated herein by reference and filed on Sep. 18, 1998).

An water-solubilizing layer is found at the outer surface of theovercoating layer. The outer layer includes a compound having at leastone linking group for attachment of the compound to the overcoatinglayer and at least one hydrophilic group spaced apart from the linkinggroup by a hydrophobic region sufficient to prevent electron chargetransfer across the hydrophobic region. The affinity for the nanocrystalsurface promotes coordination of the linking moiety to the quantum dotouter surface and the moiety with affinity for the aqueous mediumstabilizes the quantum dot suspension.

Without limitation to the scope of the present invention, the compoundmay have the formula, H_(z)X((CH₂)_(n)CO₂H)_(y) and salts thereof, whereX is S, N, P or O═P; n≧6; and z and y are selected to satisfy thevalence requirements of X. Exemplary compound for use in the inventionmay have the formula,

where X, X′ and X″ are the same or different and are selected from thegroup of S, N, P or O═P; Y is a hydrophilic moiety; and Z is ahydrophobic region having a backbone of at least six atoms. X, X′ and X″may include other substituents in order to satisfy the valencerequirements, such as for example, amines, thiols, phosphines andphosphine oxides, substituted by hydrogen or other organic moieties. Inaddition, the atoms bridging X, X′ and X″ are selected to form a5-membered to 8-membered ring upon coordination to the semiconductorsurface. The bridging atoms are typically carbon, but may be otherelements, such as oxygen, nitrogen, and sulfur. Y may be any charged orpolar group, such as carboxylates, sulfonates, phosphates, polyethyleneglycol and ammonium salt, and the like. Z is typically an alkyl group oralkenyl group, but may also include other atoms, such as carbon andnitrogen. Z may be further modified as described herein to provideattractive interactions with neighboring ligands.

In a particular preferred embodiment, the hydrophilic moiety X alsoprovides a reactive group capable of a reaction to couple the compoundto the quantum dot. For example, where the hydrophilic moiety is a —COOHor a —COO group, it may be used to couple a variety of biologicalcompounds to form a corresponding ester, amide, or anhydride. By way ofthe example only, a carboxylic acid terminated QDs can react with anamino acid to form an amide coupling. The amide may function as thecompound having affinity for a biological target.

In other preferred embodiments, a water-soluble QD is provided in whichthe outer layer has been partially substituted by a ligand whichterminates in a reactive group. The reactive group is not selected forits hydrophilic properties but rather for its ability to couple with thecompound of the invention. Exemplary reactive groups include carboxylicacid groups, thiol groups and amine groups.

In yet another embodiment of the invention, a water-soluble QD isprovided in which the outer layer is partially substituted by a ligandwhich comprises the compound of the invention. By way of example only,the compound may include a parent group terminating in a thiol, amine orphosphine or phosphine oxide, which can interact directly with thesemiconductor nanocrystal surface.

In one preferred embodiment, the present invention provides acomposition comprising a semiconductor nanocrystal that emits light at atunable wavelength and is associated with a protein. Without limitationto the scope of the invention, the protein can be a peptide or an aminoacid or derivatives thereof. Without limiting the scope of the inventionsince alternative methods may be utilized to acheive the same results,the QDs may be associated with amino acids and peptides throughconjugation of an amino acid residue with carboxylic acid groupsconjugated with N-hydroxysuccinimide (NHS) on the surface of the QDs. Ina preferred embodiment, the quantum dots are water-soluble, and asdescribed in Example 4, creating the water-soluble quantum dots involvescovering the surface of the dots with hydrophilic moieties such ascarboxylic acid groups (U.S. patent application entitled “Water-SolubleFluorescent Nanocrystals”). Carboxylic acid groups can be conjugatedwith N-hydroxysuccinimide (NHS) to activate the carbonyl group forfurther conjugation with an amino acid residue such as lysine.

As an example without limitation to the present invention, thecomposition comprises quantum dots associated with a protein that is anantibody. The antibody can be a polyclonal or a monoclonal antibody.Antibodies tagged with QDs as fluorescent markers of one color or ofmultiple colors can then be used in applications such as immunochemistryand immunocytochemistry.

As another example without limitation to the present invention, thecomposition comprises QDs conjugated to proteins with desired bindingcharacteristics such as specific binding to another protein (e.g.receptors), binding to ligands (e.g. cAMP, signaling molecules) andbinding to nucleic acids (e.g. sequence-specific binding to DNA and/orRNA).

In another preferred embodiment, the present invention provides acomposition comprising a semiconductor nanocrystal that emits light at atunable wavelength and is associated with a molecule or molecularcomplex that is capable of interacting with a biological compound. As anexample without limiting the scope of the invention, QDs can beconjugated to molecules that can interact physically with biologicalcompounds such as cells, proteins, nucleic acids, subcellular organellesand other subcellular components. For example, QDs can be associatedwith biotin which can bind to the proteins, avidin and streptavidin.Also, QDs can be associated with molecules that bind non-specifically orsequence-specifically to nucleic acids (DNA RNA). As examples withoutlimiting the scope of the invention, such molecules include smallmolecules that bind to the minor groove of DNA (for reviews, seeGeierstanger and Wemmer. Annu Rev Biophys Biomol Struct. 24:463-493,1995; and Baguley. Mol Cell Biochem. 43(3):167-181, 1982), smallmolecules that form adducts with DNA and RNA (e.g. CC-1065, seeHenderson and Hurley. J Mol Recognit. 9(2):75-87, 1996; aflatoxin, seeGarner. Mutat Res. 402(1-2):67-75, 1998; cisplatin, see Leng and Brabec.IARC Sci Publ. 125:339-348, 1994), molecules that intercalate betweenthe base pairs of DNA (e.g. methidium, propidium, ethidium, porphyrins,etc. for a review see Bailly, Henichart, Colson, and Houssier. J MolRecognit. 5(4): 155-171, 1992), radiomimetic DNA damaging agents such asbleomycin, neocarzinostatin and other enediynes (for a review, seePovirk. Mutat Res. 355(1-2):71-89, 1996), and metal complexes that bindand/or damage nucleic acids through oxidation (e.g. Cu-phenanthroline,see Perrin, Mazumder, and Sigman. Prog Nucleic Acid Res Mol Biol.52:123-151, 1996; Ru(II) and Os(II) complexes, see Moucheron, Kirsch-DeMesmaeker, and Kelly. J Photochem Photobiol B, 40(2):91-106, 1997;chemical and photochemical probes of DNA, see Nielsen, J Mol Recognit,3(1):1-25, 1990).

Molecules and higher order molecular complexes (e.g. polymers, metalcomplexes) associated with QDs can be naturally occurring or chemicallysynthesized. Molecules or higher order molecular complexes can beselected to have a desired physical, chemical or biological property.Such properties include, but are not limited to, covalent andnoncovalent association with proteins, nucleic acids, signalingmolecules, procaryotic or eukaryotic cells, viruses, subcellularorganelles and any other biological compounds. Other properties of suchmolecules, include but are not limited to, the ability to affect abiological process (e.g. cell cycle, blood coagulation, cell death,transcription, translation, signal transduction, DNA damage or cleavage,production of radicals, scavenging radicals, etc.), and the ability toalter the structure of a biological compound (e.g. crosslinking,proteolytic cleavage, radical damage, etc.). In addition, molecules andhigher order molecular complexes associated with QDs may have moregeneral physical, chemical or biological properties such as, but notlimited to, hydrophobicity, hydrophilicity, magnetism and radioactivity.

In another preferred embodiment, the present invention provides acomposition comprising a semiconductor nanocrystal that emits light at atunable wavelength and is associated with a nucleic acid. Theassociation can be direct or indirect. The nucleic acid can be anyribonucleic acid, deoxyribonucleic acid, dideoxyribonucleic acid, or anyderivatives and combinations thereof. The nucleic acid can also beoligonucleotides of any length. The oligonucleotides can besingle-stranded, double-stranded, triple-stranded or higher orderconfigurations (e.g. Holliday junctions, circular single-stranded DNA,circular double-stranded DNA, DNA cubes, (see Seeman. Annu Rev BiophysBiomol Struct. 27:225-248, 1998)).

Without limiting the scope of the present invention, QDs can beassociated with individual nucleotides, deoxynucleotides,dideoxynucleotides or any derivatives and combinations thereof and usedin DNA polymerization reactions such as DNA sequencing, reversetranscription of RNA into DNA, and polymerase chain reactions (PCR).Nucleotides also include monophosphate, diphosphate and triphophates andcyclic derivatives such as cyclic adenine monophosphate (cAMP). Otheruses of QDs conjugated to nucleic acids included fluorescence in situhybridization (FISH). In this preferred embodiment, QDs are conjugatedto oligonucleotides designed to hybridize to a specific sequence invivo. Upon hybridization, the fluorescent QD tags are used to visualizethe location of the desired DNA sequence in a cell. For example, thecellular location of a gene whose DNA sequence is partially orcompletely known can be determined using FISH. Any DNA or RNA whosesequence is partially or completely known can be visually targeted usingFISH. For example without limiting the scope of the present invention,messenger RNA (mRNA), DNA telomeres, other highly repeated DNAsequences, and other non-coding DNA sequencing can be targeted by FISH.

In another preferred embodiment, the present invention provides acomposition comprising fluorescent quantum dots associated with amolecule or reagent for detection of biological compounds such asenzymes, enzyme substrates, enzyme inhibitors, cellular organelles,lipids, phospholipids, fatty acids, sterols, cell membranes, moleculesinvolved in signal transduction, receptors and ion channels. Thecomposition also can be used to detect cell morphology and fluid flow;cell viability, proliferation and function; endocytosis and exocytosis;and reactive oxygen species (e.g. superoxide, nitric oxide, hydroxylradicals, oxygen radicals.) In addition, the composition can be used todetect hydrophobic or hydrophilic regions of biological systems.

Other applications of fluorescent markers in biological systems can befound in Haugland, R. P. Handbook of Fluorescent Probes and ResearchChemicals (Molecular Probes. Eugene, Oreg. Sixth Ed. 1996; Website,www.probes.com) which is incorporated by reference in its entirety.

In another aspect of the invention, the present invention providesmethods of detecting biological compounds using QDs. Without limitingthe scope of the present invention, the conjugation of QDs to suchmolecules as small molecules, proteins, and nucleic acids allows the useof QDs in any method of detecting the presence or amount of biologicalcompounds. Certain particular methods are discussed below in order tohighlight the advantages and utilities of the inventive compositions.These methods, include but are not limited to, fluorescenceimmunocytochemistry, fluorescence microscopy, DNA sequence analysis,fluorescence in situ hybridization (FISH), fluorescence resonance energytransfer (FRET), flow cytometry (Fluorescence Activated Cell Sorter;FACS) and diagnostic assays for biological systems.

Immunocytochemistry

Currently, fluorescence immunocytochemistry combined with fluorescencemicroscopy allows researchers to visualize biological moieties such asproteins and nucleic acids within a cell (see Current Protocols in CellBiology, John Wiley & Sons, Inc., New York; incorporated herein byreference). One method uses primary antibodies hybridized to the desiredin vivo target. Then, secondary antibodies conjugated with fluorescentdyes and targeted to the primary antibodies are used to tag the complex.The complex is visualized by exciting the dyes with a wavelength oflight matched to the dye's excitation spectrum. Fluorescent dyes thatinteract with nucleic acids such as DAPI(4′,6-diamindino-2-phenylindole), propidium iodide, ethidium bromide andHoechst dyes are used to visual DNA and RNA.

Fluorescent tags are also used to detect the presence and location ofspecific nucleic acid sequences. DNA sequences that are complementary tothe target sequences are directly labeled with fluorescent nucleotides(e.g. fluorescein-12-dUTP) and used as probes to visualize the presenceand location of the target nucleotide sequence. Examples of targetsinclude messenger RNA and genomic DNA. Alternatively, the DNA probe canbe labeled with a marker such as biotin or digoxygenin. Uponhybridization of the probe to its target sequence, afluorescent-conjugated antibody raised against the marker (e.g. biotinor digoxygenin) is used to locate and visualize the probe.

Colocalization of biological moieties in a cell is performed usingdifferent sets of antibodies for each cellular target. For example, onecellular component can be targeted with a mouse monoclonal antibody andanother component with a rabbit polyclonal antibody. These aredesignated as the primary antibody. Subsequently, secondary antibodiesto the mouse antibody or the rabbit antibody, conjugated to differentfluorescent dyes having different emission wavelengths, are used tovisualize the cellular target. In addition, fluorescent molecules suchas DAPI (4′,6-diamidino-2-phenylindole) can target and stain biologicalmoieties directly. An ideal combination of dyes for labeling multiplecomponents within a cell would have well-resolved emission spectra. Inaddition, it would be desirable for this combination of dyes to havestrong absorption at a coincident excitation wavelength.

Tunable nanocrystal quantum dots are ideal for use in fluorescenceimmunocytochemistry. The absorption spectra of QDs are broad. As aresult, a single light source (in the UV-blue region, preferably in theblue region) can be used to excite all QDs in a system simultaneously.This allows a researcher to visualize the location of all QDs (and thusthe biological components targeted) in a cell simultaneously. Inaddition, a single excitation light source simplifies the machineryinvolved in fluorescence excitation. Furthermore, the combination ofnarrow linewidths, and symmetrical, nearly gaussian lineshapes lacking atailing region in the emission spectra of QDs and the tunability of theemission wavelengths allows the use of multiple QD tags in one system.As a result, as many as 10-20 differently sized QDs, each with differenta emission spectrum, can be used simultaneously in one system and moreeasily resolved with the use of deconvolution software.

Immunoassay

One protocol for using QDs in heterogeneous immunoassays (assays inwhich the excess antibodies have to be removed in a separate step) isdescribed in FIG. 1. An antibody to an antigen is adsorbed or covalentlylinked to a solid phase (see Current Protocols in Immunology, John Wiley& Sons, Inc., New York; incorporated herein by reference). Then theantigen is added and allowed to bind to the solid-phase antibody. Afterthe excess antigen is removed, the matrix is reacted with QD-labeledantibody. After a second wash, the fluorescence can be quantified.

This protocol is amenable to multiple, parallel immunoassaying schemesas well (FIG. 2). A series of different antibodies is covalently linkedto a substrate. Then disparate antibody specific antigens can be boundto this array. Finally, different antibodies labeled with specific-sizeQDs are bound to the antigens. Again, the fluorescence from each size QDcan be quantified and the relative amount of each antigen determined.Such an extension should be possible as different sized QDs not onlyhave similar solubility properties, narrow linewidths and unique,size-dependent fluorescence frequencies, but also can be excited by thesame source of radiation (in the UV-blue, preferably in the blue regionof the spectrum).

High-Throughput DNA Sequence Analyses

QDs conjugated to nucleic acids have applications in non-cellularbiological systems. As an example without limiting the scope of theinvention, with the advent of fluorescently-labeled nucleotides,high-throughput DNA sequencing and DNA fragment analysis have becomepowerful tools in the analyses of DNA sequences (ABI system;Perkin-Elmer).

To describe these sequencing reactions briefly, four reactions areperformed to determine the positions of the four nucleotide bases withina DNA sequence. Using a DNA sample as a template, a chain of DNA issynthesized from a pool of nucleotides containing the fourdeoxynucleotides and one additional dideoxynucleotide. For example, inthe adenine sequencing reaction, DNA is synthesized from a mixture thatincludes all four deoxynucleotides (dATP, dGTP, dCTP, dTTP) plusdideoxyadenosine triphosphate (ddATP). The enzyme DNA polymerase willsynthesize the new chain of DNA by linking dNTPs. Occasionally DNApolymerase will incorporate a ddATP instead of a dATP. The ddATP in thenascent chain will then terminate the synthesis of that chain of DNA dueto the lack of the 3′ hydroxyl group as a connection to the next dNTP.Thus the DNA products from the adenine sequencing reaction will be aheterogenous mixture of DNA that vary in length with each chainterminated at a position corresponding to adenine.

The four DNA sequencing reactions are resolved by size by polyacrylamidegel electrophoresis. With singly radiolabeled (³²P or ³⁵S) DNA, the fourreactions are loaded into four individual lanes. The resolved productsof differing sizes result in a pattern of bands that indicate theidentity of a base at each nucleotide position. This pattern across thefour lanes can be read like a simple code corresponding to thenucleotide base sequence of the DNA template. With fluorescentdideoxynucleotides, samples containing all four dideoxynucleotidechain-terminating reactions can be loaded into a single lane. Resolutionof the four dideoxynucleotide reactions is possible because of thedifferent fluorescent labels for each sample. For example, ddATP can beconjugated with a green fluorescent tag. The other three ddNTP(dideoxynucleotide triphosphate) are tagged with three differentfluorescent colors. Thus, each chain-terminating ddNTP is coded with adifferent color. When all four reactions are resolved in one lane on aDNA sequencing gel, the result is one ladder of bands having fourdifferent colors. Each fluorescent color corresponds to the identity ofthe nucleotide base and can be easily analyzed by automated systems.However as previously discussed, multiple light sources are needed forexcitation of the four different fluorescent markers. The use of QDs asthe fluorescent tags for each dideoxynucleotide chain-terminatingreaction simplifies the automation of high-throughput DNA sequencingsince only a single light source is needed to excite all fourfluorescent tags.

In PCR (polymerase chain reaction)-based DNA typing and identification,short tandem repeat (STR) loci in the human genome are amplified by PCRusing primers that are labeled with fluorescent tags. The size of theseloci can differ or can coincide from person to person and depends ongenetic differences in the population. Usually multiple loci areexamined. Any locus that shows a size difference with another sampleconclusively indicates that the two samples are derived from twodifferent individuals. However, demonstrating that two samples originatefrom the same individual is less conclusive. Unlike fingerprintpatterns, the size of STR loci can coincide between two individuals.However, the statistical probability of multiple loci coinciding in sizebetween two individuals decreases as the number of loci examined isincreased. Using conventional organic fluorescent dyes, a limitation tothe number of samples resolved in a single lane (and thushigh-throughput) is the number of the fluorescent tags available and theresolution of the emission spectra. Increasing the resolution of thefluorescent tags thus would increase the capacity of the number of locitested per lane on a gel.

Fluorescence Resonance Energy Transfer (FRET)

The present invention provides a method for detecting the proximity oftwo or more biological compounds. Long-range resonance energy transferbetween QDs and between a QD and an organic fluorescent dye can occurefficiently if the spacing between them is less than approximately 100Å. This long-range effect can be exploited to study biological systems.In particular, this effect can be used to determine the proximity of twoor more biological compounds to each other. Conversely, this effect canbe used to determine that two or more biological compounds are not inproximity to each other. Advantages to using QDs combined with organicdyes for FRET include the ability to tune the narrow emission of the QDsto match precisely the excitation wavelength of organic dyes, thusreducing background signals.

In a preferred embodiment, QDs can be conjugated to a biologicalcompound or a molecule that associates with a biological compound. Afluorescent organic dye is used to label a second biological compound ora second molecule that associates with a second biological compound. TheQDs are constructed to emit light at a wavelength that corresponds tothe excitation wavelength of the organic dye. Therefore in the presenceof excitation light tuned to the excitation wavelength of the QDs andnot the dye, when a first compound labeled with QDs is in closeproximity (<100 Å) to a second compound labeled with an organic dye, theemission of the QDs will be absorbed by the dye resulting in excitationand fluorescence of the dye. Consequently, the color observed for thissystem will be the color of the fluorescent dye. If the first compoundlabeled with QDs is not in close proximity to a second compound labeledwith an organic dye that absorbs light at the wavelength emitted by theQDs, the dye will not quench the emissions of the QDs. Thus, the colorof the system will coincide with the color of the fluorescent QDs.

As an example without limiting the scope of the invention, a first DNAprobe is labeled with an organic fluorescent tag and hybridized to itstarget DNA sequence. A second DNA probe is labeled with QDs that aretuned to emit light corresponding to the excitation wavelength of theorganic fluorescent tag. If the second probe hybridizes to a targetsequence that is within at certain distance (<100 Å) to the first probe,in the presence of excitation light tuned to the QDs and not the dye,the fluorescent emission of the QDs will excite the organic dye and thusprovide a signal (color of the dye) indicating proximity of the twosequences (FIG. 3). A signal indicating a lack of close proximitybetween the two probes would be the color of the QDs since the dye wouldnot absorb the light emitted by the QDs and therefore would notfluoresce.

Alternatively, two different sized QD labels are attached to probenucleotide sequences. If these strands bind to the target DNA, then theemissions from the smaller size dots should be quenched while those fromthe larger sized ones should be enhanced. Spectroscopic quantificationof this energy transfer effect could be done in situ. Hence automateddetection of sets of DNA sequences could also be realized.

In another preferred embodiment, a method of detecting proteases usingFRET can be exploited. A peptide with a protease cleavage site issynthesized to contain a QD on one side of the cleavage site and anorganic fluorescent dye on the other side in close proximity such thatthe emission of the QD is absorbed by the dye and thus quenched. In thepresence of the protease, the peptide will be cleaved, releasing the twohalves of the peptide and removing the quenching effect of thefluorescent dye. Therefore, detection of emitted light from the QDindicates that cleavage of the peptide by the protease.

Use of QDs in Flow Cytometry/Fluorescence Activated Cell Sorter (FACS)

In this method (see Current Protocols in Cytometry and Current Protocolsin Immunology, John Wiley & Sons, Inc., New York; both of which areincorporated herein by reference), cells are labeled with a fluorescentdye and then passed, in a suspending medium, through a narrow droppingnozzle so that each cell is in a small droplet. A laser based detectorsystem is used to excite fluorescence and droplets with positivelyfluorescent cells are given an electric charge. Charged and unchargeddroplets are separated as they fall between charged plates and socollect in different tubes. The machine can be used either as ananalytical tool, counting the number of labeled cells in a population orto separate the cells for subsequent growth of the selected population.Further sophistication can be built into the system by using a secondlaser system at right angles to the first to look at a secondfluorescent label or to gauge cell size on the basis of light scatter.The utility of the method is that it looks at large numbers ofindividual cells and makes possible the separation of populations with,for example a particular surface properties.

QD technology can be applied to FACS. An advantage of using QDs in FACSis that using a single excitation light source, multiple components canbe tagged. Therefore, cells may be sorted using a variety of parameters.

Diagnostics in Biological Applications

Quantum dot technology can be used in diagnostic systems for biologicalapplications. Currently, the use of antibodies conjugated to fluorescentorganic dyes for detection of biological moieties such as white bloodcells and viruses (e.g. HIV) has limitations associated with thephysical and spectral properties of these dyes. These limitations, aspreviously discussed, include the spectral overlap observed when usingmultiple dyes with different emission spectra which contributes to thebackground when using fluorescent-conjugated antibodies as a diagnosticassay. Thus, the present invention provides a method of detectingbiological moieties as a diagnostic assay for medical purposes. In apreferred embodiment, QDs can be conjugated to molecules that are usedto detect the presence and/or concentration of a biological compound fora diagnostic assay for medical purposes.

In a preferred embodiment, QDs can be conjugated to antibodies to detectcomponents in blood or plasma such white blood cells, viruses (e.g.HIV), bacteria, cell-surface antigens of cancer cells, and anybiological component associated with human diseases and disorders. Aswith previously described biological applications of the QD technology,the use of multiple QD allows the high-throughput screening of samples.

Imaging Apparatus

The present invention also provides an apparatus for reading the outputof biological substrates encoded with multicolor fluorescent markers. Anautomated apparatus that detects multicolored luminescent biologicalsystems can be used to acquire an image of the multicolored fluorescentsystem and resolve it spectrally. Without limiting the scope of theinvention, the apparatus can detect samples by imaging or scanning.Imaging is preferred since it is faster than scanning. Imaging involvescapturing the complete fluorescent data in its entirety. Collectingfluorescent data by scanning involves moving the sample relative to amicroscope objective.

There are three parts to the apparatus: 1) an excitation source, 2) amonochromator to spectrally resolve the image, or a set of narrow bandfilters, and 3) a detector array. This apparatus can be applied tobiological systems such as individual cells, a population of cells, orwith an array of DNA.

In a preferred embodiment, for excitation of fluorescent markers, theapparatus would consist of a blue or ultraviolet light source forexcitation of the QDs. Preferably, the wavelength of the light source isshorter than the wavelength of emissions of all QDs. As an examplewithout limiting the scope of the invention since alternative methodsmay be used to obtain similar results, preferably, the light source is abroadband UV-blue light source such as a deuterium lamp with a filterattached to it. Another approach is to derive the light source from theoutput of a white light source such as a xenon lamp or a deuterium lampand pass the light through a monochromator to extract out the desiredwavelengths. Alternatively, filters could be used to extract the desiredwavelengths.

In another preferred embodiment for the excitation of fluorescentmarkers, any number of continuous wave gas lasers can be used. Theseinclude, but are not limited to, any of the argon ion laser lines (e.g.457, 488, 514 nm, etc.) or a HeCd laser. Furthermore, solid state diodelasers that have an output in the blue region of the spectrum such asGaN-based lasers or GaAs-based lasers with doubled output could be used.In addition, YAG or YLF-based lasers with doubled or tripled output, orany pulsed laser with an output also in the blue region can be used.

In a preferred embodiment, for the spectral resolution of thefluorescent QDs in a system, preferably the luminescence from the QDs ispassed through an image-subtracting double monochromator. An alternativemethod of resolving the spectra of each QD in a system with multiple QDsis to pass the luminescent light through two single monochromators withthe second one reversed from the first. The double monochromatorconsists of two gratings or two prisms and a slit between the twogratings. The first grating spreads the colors spatially. The slitselects a small band of colors and the second grating recreates theimage. This image contains only the colors specific to the output of aQD of a particular size (emission).

In another preferred embodiment for resolving the emission spectra of asystem containing multiple QDs is to use a computer-controlled colorfilter wheel where each filter is a narrow band filter centered at thewavelength of emission of one of the QDs in a system.

In a preferred embodiment, the fluorescent images are recorded using acamera preferably fitted with a charge-coupled device. Anytwo-dimensional detector can be used. Software is then used to color theimages artificially to the actual wavelengths observed. The system thenmoves the gratings to a new color and repeats the process. The finaloutput consists of a set of images of the same spatial region, eachcolored to a particular wavelength. This provides the necessaryinformation for rapid analysis of the data.

In another preferred embodiment, an alternative method of detecting thefluorescent QDs in biological systems is to scan the samples. Anapparatus using the scanning method of detection collects luminescentdata from the sample relative to a microscope objective by moving eitherthe sample or the objective. The resulting luminescence is passedthought a single monochromator, a grating or a prism to resolve thecolors spectrally. Alternatively, filters could be used to resolve thecolors spectrally.

For the scanning method of detection, the detector is a diode arraywhich records the colors that are emitted at a particular spatialposition. Software then recreates the scanned image, resulting in asingle picture (file) containing all the colors of the QDs in thesample.

Since an entire spectrum is captured in a single file, in systems withmultiple QDs, spectral deconvolution is necessary and easily performedto resolve overlapping spectra. As previously discussed, the narrowspectral linewidths and nearly gaussian symmetrical lineshapes lacking atailing region observed for the emission spectra of QDs combined withthe tunability of the emission wavelengths of QDs allows high spectralresolution in a system with multiple QDs. In theory, up to 10-20different-sized QDs from different preparations of QDs, with each samplehaving a different emission spectrum, can be used simultaneously in onesystem with the overlapping spectra easily resolved using deconvolutionsoftware.

Photoluminescence of Single Nanocrystal Quantum Dots

Single nanocrystal quantum dots have detectable luminescence (Nirmal etal. Nature 383: 802, 1996; and Empedocles et al. Phys. Rev. Lett.77:3873, 1996; both incorporated herein by reference) which can beapplied to biological systems. An advantage of having highly fluorescentsingle QDs that are detectable and associated with biological compoundsis that this allows the detection of very small quantities of biologicalmolecules. Thus, the throughput of assays that screen large numbers ofsamples can be improved by utilizing single QDs associated withbiological compounds to decrease the sample size, and consequentlyallowing a greater number of samples to be screen at any one time.

The following Examples illustrate the preferred modes of making andpracticing the present invention, but are not meant to limit the scopeof the invention since alternative methods may be used to obtain similarresults.

EXAMPLES Example 1 Preparation of TOPO Capped-(CdSe)ZnS

(a) Preparation of CdSe Trioctylphosphine oxide (TOPO, 90% pure) andtrioctylphosphine (TOP, 95% pure) were obtained from Strem and Fluka,respectively. Dimethyl cadmium (CdMe₂) and diethyl zinc (ZnEt₂) werepurchased from Alfa and Fluka, respectively, and both materials werefiltered separately through a 0.2:m filter in an inert atmosphere box.Trioctylphosphine selenide was prepare by dissolving 0.1 mols of Se shotin 100 ml of TOP thus producing a 1M solution of TOPSe.Hexamethyl(disilathiane) (TMS₂S) was used as purchased from Aldrich.HPLC grade n-hexane, methanol, pyridine and n-butanol were purchasedfrom EM Sciences.

The typical preparation of TOP/TOPO capped CdSe nanocrystals follows.TOPO (30 g) was placed in a flask and dried under vacuum (˜1 Torr) at180° C. for 1 hour. The flask was then filled with nitrogen and heatedto 350° C. In an inert atmosphere drybox the following injectionsolution was prepared: CdMe₂ (200 microliters, 2.78 mmol), 1 M TOPSesolution (4.0 mL, 4.0 mmol), and TOP (16 mL). The injection solution wasthoroughly mixed, loaded into a syringe, and removed from the drybox.

The heat was removed from the reaction flask and the reagent mixture wasdelivered into the vigorously stirring TOPO with a single continuousinjection. This produces a deep-yellow/orange solution with a sharpabsorption feature at 470-500 nm and a sudden temperature decrease to˜240° C. Heating was restored to the reaction flask and the temperaturewas gradually raised to 260-280° C.

Aliquots of the reaction solution were removed at regular intervals(5-10 min) and absorption spectra taken to monitor the growth of thecrystallites. The best samples were prepared over a period of a fewhours steady growth by modulating the growth temperature in response tochanges in the size distribution, as estimated from the sharpness of thefeatures in the absorption spectra. The temperature was lowered 5-10° C.in response to an increase in the size distribution. Alternatively, thereaction can also be stopped at this point. When growth appears to stop,the temperature is raised 5-10° C. When the desired absorptioncharacteristics were observed, the reaction flask was allowed to cool to˜60° C. and 20 mL of butanol were added to prevent solidification of theTOPO. Addition of a large excess of methanol causes the particles toflocculate. The flocculate was separated from the supernatant liquid bycentrifugation; the resulting powder can be dispersed in a variety oforganic solvents (alkanes, ethers, chloroform, tetrahydrofuran, toluene,etc.) to produce an optically clear solution.

(b) Preparation of (CdSe)ZnS A flask containing 5 g of TOPO was heatedto 190 EC under vacuum for several hours then cooled to 60 EC afterwhich 0.5 mL trioctylphosphine (TOP) was added. Roughly 0.1-0.4:mols ofCdSe dots dispersed in hexane were transferred into the reaction vesselvia syringe and the solvent was pumped off.

Diethyl zinc (ZnEt₂) and hexamethyldisilathiane ((TMS)₂S) were used asthe Zn and S precursors, respectively. The amounts of Zn and Sprecursors needed to grow a ZnS shell of desired thickness for each CdSesample were determined as follows: First, the average radius of the CdSedots was estimated from TEM or SAXS measurements. Next, the ratio of ZnSto CdSe necessary to form a shell of desired thickness was calculatedbased on the ratio of the shell volume to that of the core assuming aspherical core and shell and taking into account the bulk latticeparameters of CdSe and ZnS. For larger particles the ratio of Zn to Cdnecessary to achieve the same thickness shell is less than for thesmaller dots. The actual amount of ZnS that grows onto the CdSe coreswas generally less than the amount added due to incomplete reaction ofthe precursors and to loss of some material on the walls of the flaskduring the addition.

Equimolar amounts of the precursors were dissolved in 2-4 mL TOP insidean inert atmosphere glove box. The precursor solution was loaded into asyringe and transferred to an addition funnel attached to the reactionflask. The reaction flask containing CdSe dots dispersed in TOPO and TOPwas heated under an atmosphere of N₂. The temperature at which theprecursors were added ranged from 140° C. for 23 Å diameter dots to 220°C. for 55 Å diameter dots. When the desired temperature was reached theZn and S precursors were added dropwise to the vigorously stirringreaction mixture over a period of 5-10 minutes.

After the addition was complete the mixture was cooled to 90° C. andleft stirring for several hours. Butanol (5 mL) was added to the mixtureto prevent the TOPO from solidifying upon cooling to room temperature.The overcoated particles were stored in their growth solution to ensurethat the surface of the dots remained passivated with TOPO. They werelater recovered in powder form by precipitating with methanol andredispersing into a variety of solvents including hexane, chloroform,toluene, THF and pyridine.

Example 2 Preparation of a Water-Soluble Quantum Dots Using Long ChainMercaptocarboxylic Acid

TOPO capped-(CdSe)ZnS quantum dots were prepared as described inExample 1. The overcoated (CdSe)ZnS dots were precipitated from thegrowth solution using a mixture of butanol and methanol. To obtain theprecipitated quantum dots, the solution was centrifuged for 5-10 min,the supernatant was decanted and the residue was washed with methanol(2×).

The residue was weighed. The weight of the TOPO cap was assumed to be30% of the total weight; and a 30-fold molar excess of the new cappingcompound, 11-mercaptoundecanoic acid (MUA) was added. The residue andMUA (neat solution) were stirred at 60° C. for 8-12 hours. A volume oftetrahydrofuran (THF) equal to the added MUA was added to the MUA/dotmixture, with the mixture was still hot. A clear solution resulted andthe coated quantum dots were stored under THF.

The coated quantum dots are rendered water-soluble by deprotonation ofthe carboxylic acid functional group of the MUA (FIG. 4). Thedeprotonation was accomplished by adding a suspension of potassiumt-butoxide in THF to the MUA-quantum dot/THF solution. A gel resulted,which was then centrifuged and the supernatant liquid was poured off.The residue was washed twice with THF, centrifuged each time and thesupernatant liquid poured off. The final residue was allowed to dry inair for 10 minutes. Deionized water (Millipore) was added to the residueuntil a clear solution formed.

The resultant coated quantum dots were tested for photoluminescentquantum yield. A CdSe quantum dot with a four monolayer coating of ZnScoated as described had an absorption band a 480 nm and aphotoluminescent band at 500 nm, with a quantum yield of 12%. A secondCdSe quantum dot with a four monolayer coating of ZnS coated asdescribed had an absorption band a 526 nm and a photoluminescent band at542 nm, with a quantum yield of 18%.

Example 3 Associating a Water-Solubilzed Quantum Dot with a Protein

CdSe quantum dots overcoated with ZnS were synthesized, purified, andsolubilized in water as previously described. Samples used in thisexperiment had 40 Å diameter CdSe cores, a ZnS shell which was nominally4 monolayers (about 9 Å) thick, and capped with 11-mercaptoundecanoicacid (MUA).

The following three reagents were mixed: 5.8 mg of1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC), 2.4mg of N-hydroxysuccinimide (NHS), and 4 mL of a 0.82 micromolar solutionof avidin in Millipore filtered water. The initially acidic mixture wastreated with 0.1 M NaOH (aq) to adjust the pH to 7.6. Then 3 mL of a 2.1micromolar aqueous solution of (CdSe)ZnS quantum dots was added. Themixture was stirred for 1 hour at room temperature. Excess reagents werequenched with 1 drop of 0.25 M ethanolamine in water.

To determine whether avidin coupling was successful, the coloredreaction solution was passed through a short column containingbiotin-coated acrylic beads. The filtrate which emerged was nearlycolorless. The column was then washed with 10-fold volume of water.Under excitation with ultraviolet light, the beads displayed strongfluorescence due to the bound quantum dots, indicating successfulcoupling to avidin. A control experiment using only quantum dots andavidin with reagents to couple them (i.e. no EDAC or NHS) produced beadswith little or no fluorescence, confirming that without avidin-couplingthe quantum dots do not bind to the biotin coated beads.

Example 4 Biotin Hexane Dithiol (BHDT) Formation

This procedure exploits the activated carboxylic acid group present inthe biotin derivative, biotin-N-hydroxysuccinimide (BNHS; PierceChemicals, Rockford, Ill.) to made a biotin derivative which terminatesin a thiol (SH) group (FIG. 5). The presence of a thiol group is desiredbecause thiols, in general, tend to adsorb to metal surfaces. Therefore,the thiol linkage can be exploited to attach biotin to the water-solublequantum dots.

BNHS was dissolved in DMF and a 10-fold excess of 1,6-hexanedithiol wasadded in the presence of a weak base (triethylamine). The solution wasstirred at room temperature for 16 hours. An NHS precipitate results andthe solution was filtered to remove this NHS precipitate. Theprecipitate was washed with DMF. The precipitate was reduced to aminimum volume by removing solvent under a vacuum. Ether was then addedto the concentrated solution to precipitate crude product. The productwas isolated by filtration and the residue was pumped under a vacuum toremove the excess dithiol. A white powder (BHDT) was isolated and storedin the glove-box refrigerator to prevent thiol oxidation into disulfide.The resultant yield was approximately 68%.

Example 5 Biotin-Amine Formation

The philosophy of this procedure is similar to the one described inExample 6. In this example, the activated carboxylic group in biotin isutilized to make a biotin derivative with a terminal amine group (FIG.6). As with thiols, amines conjugate to metal surfaces and can be usedto attach biotin to the dots.

100 mg of BNHS was added to 2 ml DMF in a vial and mixed until all theBNHS had dissolved. Next, 0.9 ml of 1,3 diaminopropane (a 30 foldexcess) was added to another vial. The BNHS/DMF solution was pipettedinto the vial containing the neat 1,3-diaminopropane in 2 aliquots. Theadditions were performed in approximately 2 minutes and were spaced by 5minutes. The resulting solution was stirred at room temperature for 24hours, and a white precipitate (NHS) was formed. The NHS precipitate wasremoved by centrifuging, and the clear supernatant was transferred toanother vial. Excess ether was added to the supernatant. Upon shaking,an immiscible layer was formed at the bottom which was transferred to around-bottomed flask. DMF and excess diamine were then removed undervacuum to yield a white powder. The resultant yield was approximately72%.

Example 6 Biotin-Thiol-Dot Complex Formation

The aim of this protocol is to attach a biotin cap onto the surface ofthe dots. The thiol end group of BHDT should adsorb to the dot surface(FIG. 7). Excess MUA from the cap was removed from the quantum dot/THFsolution by precipitating the dots with a hexane/butanol mixture. Theprecipitate was redispersed in DMF and then precipitated again with ahexane/BuOH mixture. The precipitate was allowed to dry in air for 20-25minutes, weighed, and redissolved in DMF. To calculate the amount ofBHDT to dissolve in DMF, it was estimated that 30% of the total weightof the dot was derived from the cap. With that estimation, a 10-foldexcess of BHDT (relative to the cap) was dissolved in DMF in a separatevial. The BHDT solution was then added to the QD/DMF solution over a 5minute period. This mixture was stirred at room temperature forapproximately 15 hours. The reaction was stopped by centrifugation,saving only the supernatant. A solution of potassium tert-butoxide inDMF was used to deprotonate the MUA acid cap. A colored precipitate wasformed which is the water-soluble product. This mixture was subjected tocentrifugation and the clear supernatant was discarded. Nophotoluminescence was observed from this layer indicating that all QDswere successfully precipitated out of solution.

The precipitate was dissolved in deionized H₂O (Millipore; Bedford,Mass.). The resulting solution was filtered through a 0.2 μm filter(Millipore), and transferred to a Ultrafree-4™ concentrator (Millipore).The solution was spun three times through the concentrator, and aftereach spin, the tubes were topped off with water. The concentratedsolution was transferred to a vial and diluted with water. To confirmthat biotin was successfully conjugated to the dots, the resultingsolution was passed over an immobilized avidin column (Ultra-link™,Pierce, Rockford, Ill.). Dots derivatized with biotin were retained bythe column, resulting in the fluorescence of the column when illuminatedwith a UV-lamp. Control columns, which had non-biotinylated dots passedover them, showed no fluorescence when illuminated with a UV lamp

Example 7 Biotin-Amine-Dot Complex Formation

This protocol allows one to attach biotin to the surface of the dots.Conjugation of biotin to the dots is achieved through the primary aminegroup at the end of biotin. Again, the affinity of the amine group forthe surface of the dot is being exploited in this protocol (FIG. 8).

In this protocol, the MUA capped dots were precipitated from the QD/THFsolution using a hexane/BuOH mixture. The dots were air-dried forapproximately 5 minutes and weighed. Deprotonation of the dots wasaccomplished by adjusting the pH of the solution to 10.5 with a 1Msolution of NH₄OH. To calculate the amount of excess biotin-amine touse, it was estimated that 30% of the overall weight of the dots wasderived from the cap. As such, a 10-fold excess (to the cap) of thebiotin-amine reagent that was previously synthesized as in Example 5 wasweighed out in a separate vial. This biotin derivative was thendissolved in a minimum volume of water. The solution containing thebiotin-amine conjugate was pipetted into the solution of deprotonateddots over the course of about 3 minutes, and then stirred at roomtemperature for approximately 12 hours. The reaction was stopped bycentrifugation, and the resulting supernatant was passed through a 0.2μm filter (Millipore).

After filtration, the solution was transferred to a Ultrafree-4™concentrator (Millipore; MW cutoff=30 kDa). The solution was spun threetimes through the concentrator, and after each spin, the tubes weretopped off with deionized water. The final concentrated solution wasdiluted again with water and refiltered through a 0.2 μm filter. Theresulting clear solution was passed over an immobilized avidin column(Ultra-link™ matrix; Pierce) to confirm biotinylation of the dots asdescribed in Example 6.

Example 8 Biotin-Amine-Dot Complex Formation (Alternate Route)

Unlike the procedures described in the previous Examples, this protocolutilizes the carboxylic acid groups that cap the surface of thewater-soluble dots described in Example 2 (see FIG. 9). An amide bond isformed by conjugating a biotin-primary amine derivative to thecarboxylic acid group at the surface of the dots. This coupling is donewith the aid of 1-ethyl-3-(3-dimethylaminopropyl) carboimidehydrochloride (EDC; Pierce Chemicals, MW=191.7 g/mol), another groupthat activates the carboxylic acid group for use in subsequentreactions.

The MUA-capped dots dissolved in THF were precipitated by deprotonatingthe carboxylic acid group. This deprotonation was accomplished by addinga potassium tert-butoxide/THF suspension. The resulting residue waswashed with THF twice, air-dried for 10-15 minutes, and weighed.Deionized water was then added to the residue and the suspension wasshaken until the residue was completely dissolved. An aliquot from thissolution was then concentrated by centrifugation three times using anUltrafree-4™ concentrator (Millipore). Again, after each concentration,the tube was topped off with Millipore filtered water. The pH of thesolution was adjusted to 9 using a 1M solution of NH₄OH.

For the following calculations, the weight of the acid cap was assumedto be 30% of the total weight of the dots. A solution of EZ-Link™biotin-PEO-LC-Amine (Pierce Chemicals, MW=418 g/mol) and(1-ethyl-3-(3-dimethylaminopropyl) carboimide hydrochloride (EDC) inwater at a molar equivalent of 1:1 (biotin derivative:acid cap) and 10:1(EDC:acid cap) was then added to the dots (pH=8.5). This mixture wasstirred at room temperature for 2-3 hours. The reaction was stopped byfiltering the solution through a 0.2 μm Millipore filter twice.

As in Example 6, conjugation of biotin to the dots was confirmed bypassing the sample over an avidin column. Successful conjugationresulted in a fluorescent column. A control column with non-biotinylateddots passed over it did not fluoresce.

Example 9 Quantum Dot-Oligonucleotide Complex Formation

This procedure is derived from the synthesis of the biotin-amine-dotcomplex. In particular, molar equivalents used in Example 5 will be usedto complex the quantum dots to 5′ amine-labeled oligonucleotides.

A solution of MUA-capped dots dissolved in THF will be deprotonatedusing potassium tert-butoxide. The resulting gel will be washed with THFtwice, centrifuged, and the subsequent supernatant discarded. The finalresidue is air-dried and weighed. Deionized water is added to the driedresidue and shaken until a clear solution results. An aliquot of thesolution is desalted and concentrated twice using an Ultrafree-4™concentrator (Millipore). After each concentration, the concentratortube is topped off with deionized water.

The amount of dots is estimated from the ratio of volumes of the aliquotand the total volume of water used. Relative to the amount of dots, onemolar equivalent of 5′ amine-labeled oligonucleotide and 10 molarequivalents of EDC (Pierce, mol wt=192) are dissolved in water. The pHof this solution is adjusted to 8.5. This solution is then added to thesolution of dots described in the preceding section and stirred at roomtemperature for 2-3 hours. The reaction is stopped by passing thesolution through 0.2 μm Millipore filter, and concentrating the filtrateusing an Ultrafree-4™ concentrator.

Conjugation of the dots to the oligonucleotide will be checked using aprotocol described in the next Example.

Example 10 Quantum Dot-Oligonucleotide Complex Formation Check

The same column used to confirm biotin-dot formation can be modified tocheck for oligo-dot complex formation. A solution of 5′ biotin-labeledoligonucleotide, complementary in sequence to the oligonucleotidecomplexed with the dots, will be passed through an Ultra-link™ (PierceChemicals) immobilized avidin column. The biotin will bind to the avidinto form an immobilized, oligonucleotide column. The oligonucleotide-dotconjugation will then be checked by passing the solution of theoligonucleotide-dot complex over this column. Complementary DNAsequences will be allowed to hybridize at the appropriate hybridizationtemperature for 12 hours as calculated by standard methods (Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989; incorporated herein byreference). After hybridization, the column will be washed with water toremove any unbound oligonucleotide-dots complexes. Successfuloligonucleotide-dot conjugation and subsequent hybridization to thecomplementary oligonucleotide column should result in a column thatfluoresces with the appropriate excitation light. No fluorescencesuggests all the color disappeared upon elution and that no complex wasformed between the dots and the oligonucleotide.

1. A method of detecting biological moieties comprising: providing aplurality of compositions capable of characteristic spectral emissions,the composition comprising a compound and a semiconductor nanocrystalassociated with the compound, wherein each of the members of theplurality is characterized in that: the nanocrystal of the member of theplurality has an emission spectrum distinct from the other members ofthe plurality and a quantum yield of greater than 10% in water, and thecompound of the member of the plurality has a corresponding biologicalmoiety distinct from other biological moieties in the sample; allowing asample containing or suspected of containing one or more biologicalmoieties to interact with the compositions; and monitoring the spectralemission of each interaction between each composition and eachbiological moiety of the sample, wherein the interaction between thebiological moiety and the composition comprises a noncovalentinteraction.
 2. The method of claim 1, wherein the compound comprises amolecular complex with a molecule associated with the nanocrystalcomplexed to a second molecule that interacts with the biologicalmoiety.
 3. The method of claim 1, wherein each interaction between eachcomposition and each biological moiety of the sample are monitoredsubstantially simultaneously.
 4. The method of claim 1, wherein thespectral emission provides information about a biological state orevent.
 5. The method of claim 4, wherein the spectral emission providesinformation about the amount of biological moiety in the sample.
 6. Themethod of claim 4, wherein the spectral emission provides informationabout the presence of the biological moiety in the sample.
 7. The methodof claim 1, wherein the semiconductor nanocrystal is water-soluble. 8.The method of claim 1, wherein the semiconductor nanocrystal comprises acore comprising a semiconductor material, and a layer overcoating thecore comprising a semiconductor material.
 9. The method of claim 1,wherein the spectral emission is tunable to a desired wavelength bycontrolling the size of the nanocrystal.
 10. The method of claim 1,wherein monitoring the spectral emission occurs in assays selected fromthe group consisting of: immunochemistry, immunocytochemistry,immunobiology, immunofluorescence, DNA sequence analysis, fluorescenceresonance energy transfer, flow cytometry, fluorescence activated cellsorting, diagnostics in biological systems, and high-throughputscreening.
 11. The method of claim 1, wherein the noncovalentinteraction comprises hydrophobic interaction, hydrophilic interaction,electrostatic interaction, van der Waals interaction, or magneticinteraction.
 12. The method of claim 1, wherein the biological moietycomprises a small molecule.
 13. The method of claim 1, wherein thebiological moiety comprises a protein, peptide or antibody.
 14. Themethod of claim 1, wherein the biological moiety comprises a nucleicacid.
 15. The method of claim 14, wherein the nucleic acid comprises DNAor RNA.
 16. The method of claim 1, wherein the biological moietycomprises an amino acid.
 17. The method of claim 1, wherein thebiological moiety comprises a ligand.
 18. The method of claim 1, whereinthe biological moiety comprises an antigen.
 19. The method of claim 1,wherein the biological moiety comprises a cell.
 20. The method of claim1, wherein the biological moiety comprises a subcellular organelle. 21.A method of detecting biological moieties comprising: providing aplurality of compositions capable of characteristic spectral emissions,the composition comprising a compound and a semiconductor nanocrystalassociated with the compound, wherein each of the members of theplurality is characterized in that: the nanocrystal of the member of theplurality has an emission spectrum distinct from the other members ofthe plurality, and the compound of the member of the plurality has acorresponding biological moiety distinct from other biological moietiesin the sample and is associated with the nanocrystal by a ligand havingat least one linking group for attachment to the nanocrystal spacedapart from a hydrophilic group by an alkyl or alkenyl group; allowing asample containing or suspected of containing one or more biologicalmoieties to interact with the compositions; and monitoring the spectralemission of each interaction between each composition and eachbiological moiety of the sample, wherein the interaction between thebiological moiety and the composition comprises a noncovalentinteraction.
 22. The method of claim 21, wherein the hydrophilic groupis selected from the group consisting of carboxylic acid, carboxylate,sulfonate, hydroxide, alkoxide, ammonium, phosphate, and phosphonate.23. The method of claim 21, wherein each interaction between eachcomposition and each biological moiety of the sample are monitoredsubstantially simultaneously.
 24. The method of claim 21, wherein thespectral emission provides information about a biological state orevent.
 25. The method of claim 21, wherein the semiconductor nanocrystalis water-soluble.
 26. The method of claim 21, wherein the semiconductornanocrystal comprises a core comprising a semiconductor material, and alayer overcoating the core comprising a semiconductor material.
 27. Themethod of claim 21, wherein the biological moiety comprises a smallmolecule.
 28. The method of claim 21, wherein the biological moietycomprises a protein, peptide or antibody.
 29. The method of claim 21,wherein the biological moiety comprises a nucleic avid.
 30. The methodof claim 29, wherein the nucleic acid comprises DNA or RNA.
 31. Themethod of claim 21, wherein the biological moiety comprises an aminoacid.
 32. The method of claim 21, wherein the biological moietycomprises a ligand.
 33. The method of claim 21, wherein the biologicalmoiety comprises an antigen.
 34. The method of claim 21, wherein thebiological moiety comprises a cell.
 35. The method of claim 21, whereinthe biological moiety comprises a subcellular organelle.
 36. The methodof claim 21, wherein the spectral emission is tunable to a desiredwavelength by controlling the size of the nanocrystal.
 37. The method ofclaim 21, wherein monitoring the spectral emission occurs in assaysselected from the group consisting of: immunochemistry,immunocytochemistry, immunobiology, immunofluorescence, DNA sequenceanalysis, fluorescence resonance energy transfer, flow cytometry,fluorescence activated cell sorting, diagnostics in biological systems,and high throughput screening.
 38. The method of claim 21, wherein thespectral emission is tunable to a desired wavelength by controlling thesize of the nanocrystal.
 39. The method of claim 21, wherein monitoringthe spectral emission occurs in assays selected from the groupconsisting of: immunochemistry, immunocytochemistry, immunobiology,immunofluorescence, DNA sequence analysis, fluorescence resonance energytransfer, flow cytometry, fluorescence activated cell sorting,diagnostics in biological systems, and high throughput screening. 40.The method of claim 21, wherein the noncovalent interaction compriseshydrophobic interaction, hydrophilic interaction, electrostaticinteraction, van der Waals interaction, or magnetic interaction.